1. Field of the Invention
The present invention relates to methods of using probes and microarrays to measure multiple different stable isotopes in nucleic acids and identification and analysis of microbial communities.
2. Related Art
Identification of microorganisms responsible for specific metabolic processes remains a major challenge in environmental microbiology, one that requires the integration of multiple techniques.
Nucleic acid stable isotope probing (SIP) techniques (5, 6) are currently the most widely used means to directly connect specific substrate utilization to microbial identity, a grand challenge in the field of microbial ecology (7). For traditional SIP, natural microbial communities are incubated in the presence of a substrate enriched in a rare stable isotope (either 13C or 15N). The organisms, including their nucleic acids, incorporate the substrate and become isotopically enriched over time. Ultracentrifugation is used to separate isotopically enriched nucleic acids from lighter, unenriched nucleic acids for molecular analysis. In the past decade, these approaches have generated many advances in the understanding of microbial bioremediation, plant-microbe interactions and food web dynamics (8), yet they remain hindered by logistical drawbacks (9). These issues are intensified when working with density-gradient centrifugation of RNA, where the focus is on active organisms that are not necessarily replicating. Most notably, traditional DNA- and RNA-SIP isotope exposure risks fertilization effects by requiring high substrate concentrations in order to meet the sensitivity threshold of density gradient separation (in many systems>20% 13C DNA) (10) and is extremely difficult to perform with 15N labeled substrates (>40% 15N DNA required) (11). Other disadvantages include long exposure times (risking community cross-feeding), low-throughput (1-2 weeks lab processing time per sample batch), and incomplete quantification. Though related culture-independent approaches also have ideal qualities such as high sensitivity or in situ resolution (e.g. 13C-PLFA (12); EL FISH (13), FISH MAR (14), isotope arrays (15)), none combines high throughput, sensitivity, taxonomic resolution, and quantitative estimates of multiple stable isotope (15N and 13C) incorporation.